Infinite 200 pro reader

Cell viability was assessed by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich, Inc.). MTT was solubilized in culture media without FCS at the final concentration of 0.5 mg/mL. Cells were incubated with MTT for 50 min at +37°C in the dark. The purple formazan crystals produced by viable cells were dissolved by DMSO and the absorbance was quantified at 550 nm with an Infinite^® 200 Pro reader (Tecan Trading AG).

Precultures were grown over night in 10 ml LB medium inoculated from a single colony. From a preculture 10 ml of LB medium was inoculated with an OD₆₀₀ = 0.1 in triplicates and cultivated at 30 °C. At defined time points, OD₆₀₀ was measured and 100 µl of each culture were transferred into the well of a microtiter plate (corning 96 flat bottom white, clear bottom polystyrol, -pure Grade™ S-, Ref: 781,670, BRANDplates^®). Bioluminescence was measured with an Infinite 200 PRO reader (Tecan Trading AG, Männedorf. Switzerland) (Shaking linear duration: 4 s, shaking linear amplitude: 1 mm, top reading, mode: luminescence, attenuation: none, integration time: 1,000 ms, settle time: 0 ms). For comparability, bioluminescence was normalized by division through the OD₆₀₀ measured at the same time point. GraphPad Prism 7.00 (GraphPad Software, Inc, La Jolla, CA, USA) was used for calculating P values (unpaired t-test).

The ability to form biofilms on a plastic surface was monitored using a slightly modified version of the microtiter plate biofilm assay published by Merritt, Kadouri & O’Toole (2005) (link). Cells of an overnight culture grown in LB medium were collected with centrifugation (2 min, 10,000× g, RT) and resuspended in Schneider’s insect medium adjusting to an OD₆₀₀ = 0.6. For every strain, 100 µl was inoculated in six replicate wells (Polystyrene (PS) Microtest Plate 96 Well.R, round bottom, Ref 82.1582.001; Sarstedt, Nümbrecht, Germany) and incubated for 72 h at 30 °C in a humidified box. We cultivated the strains in Schneider’s insect medium, when performing the biofilm assay. The wells were washed twice with H₂O and biofilms were stained with 0.1% crystal violet solution (solved in H₂O) for 10 min. Unbound dye was removed and the stained biofilms were air dried. The amount of biofilm bound crystal violet serves as a measure for biofilm formation. Dye was dissolved using 30% acetic acid, with 100 µl of this solution transferred to a new microtiter plate (Polystyrene (PS) Microtest Plate 96 Well.F, flat bottom, Ref 82.1581.001; Sarstedt, Nümbrecht, Germany) for measuring the absorption at a wavelength of 570 nm using a microplate reader (Infinite 200 PRO reader; Tecan Trading AG, Männedorf. Switzerland).

Cell viability was performed by MTT assay as previously described (Lefebvre et al., 2020). Cells were seeded in 6-well plates in double of density as invasion assays at the rate of 8 × 10⁴ cells per well. After 24 h of incubation, the medium is replaced by 800 µl of MTT (tetrazolium salts of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma-Aldrich, Darmstadt Germany) solubilized in culture medium without FBS at 0.5 mg/ml in each well. Plates were then incubated for 50 min at 37°C in obscurity. To dissolve purple formazan crystals formed by living cells, the culture medium was replaced with DMSO (dimethyl sulfoxide, Sigma-Aldrich, Darmstadt Germany). Absorbance of each well was quantified at 550 nm using an Infinite® 200 Pro Reader (Tecan Trading AG, Männedorf, Switzerland).

Patients´ sera were obtained during surgery and immediately stored at −80 °C. Likewise, healthy serum samples were immediately stored at −80 °C after removal. Total protein concentration was measured using Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific Inc, Rockford, IL, USA) and Infinite 200® PRO Reader (Tecan Group Ltd., Männedorf, Switzerland) according to the manufacturer’s protocol. Magellan™ Data Analysis Software (Tecan Group Ltd., Männedorf, Switzerland) was taken for analysis of these data. All serum samples were diluted to a concentration of 0.1 μg/μl of total protein.